The discovery of presenilin (PS) as the first intramembrane aspartyl protease and the catalytic center of gamma-secretase occurred in Project 1. Since then, >100 substrates have been identified. PS/gamma-secretase mediates critical signaling pathways necessary for life in all metazoans, and its cleavage of APP releases the amyloid B-protein that accumulates in all patients with AD. After identifying PS as a protease, Project 1 has continued to contribute actively to PS biology. We proposed - and provided the first evidence - that holoPS undergoes autocatalytic endoproteolysis to generate the active heterodimer, first reconstituted PS and its 3 cofactors in mammalian cells, purified the protease to homogeneity, obtained the first 3D structure of the ycomplex by EM, conducted SILAC screens to identify several new substrates, and designed many gamma-secretase inhibitors, some of which are potent and much more APP-selective than compounds tried in humans. The Project is now revised to respond to all of the SEP's helpful critiques. We propose to study 4 related topics in the biochemistry of gamma-secretase. 1: A new cell biological model of secretase processing We have discovered that contrary to current concepts, alpha- (ADAM 10), beta- and gamma-secretases exist in part in a large protein complex that can mediate efficient sequential processing of substrates. Our extensive supporting data include robust co-IP of endogenous a- and gamma-secretases from wt brain and the sequential alpha/gamma processing of an APP substrate. We propose to fully confirm this new model of regulated intramembrane proteolysis and ask if it generalizes to another membrane protease pair: S1P/S2P. 2: The complex regulation of gamma-secretase by membrane lipids. We will extend our recent evidence that certain head groups and fatty acyl side chains of membrane lipids potently up- and down-regulate gamma-cleavages, including the key A(i42/40 ratio. We'll seek to validate robust in vitro effects of certain lipids by manipulating their cognate biosynthetic and catabolic enzymes in vivo. 3: Toward greater structural resolution of the gamma-secretase complex Working with leading structural biologists, we will pursue the technically challenging but essential quest for the structure of gamma-secretase via: a) further cryo-EM analyses of 2D crystals; b) 3D x-ray crystallography of individual gamma-components (PS, Net, Pen-2); and c) attempted 3D x-ray crystallography of the purified holo-enzyme. 4: Refining potent and selective Notch-sparing gamma-inhibitors and defining their mechanism. Building on more than 1,600 compounds we've synthesized, we will develop SARs for inhibiting APP vs. Notch, and for the most selective compounds, assess cleavage of other gamma-substrates and test them in mice. These aims build on our experience to tackle some of the thorniest problems in gamma-secretase biology.